Anti-erbb2 antibodies

ABSTRACT

Anti-ErbB2 antibodies are described which bind to an epitope in Domain 1 of ErbB2 and induce cell death via apoptosis. Various uses for these antibodies are also described.

BACKGROUND OF THE INVENTION

This is a non-provisional application filed under 37 CFR 1.53(b)(1),claiming priority under USC Section 119(e) to provisional ApplicationSer. No. 60/028,811, filed on 18 Oct., 1996.

1. Field of the Invention

This invention relates generally to antibodies which bind the ErbB2receptor. In particular, it pertains to anti-ErbB2 antibodies which bindto an epitope in Domain 1 of ErbB2 and induce cell death via apoptosis.

2. Description of Related Art

Transduction of signals that regulate cell growth and differentiation ismodulated in part by phosphorylation of various cellular proteins.Protein tyrosine kinases are enzymes that are involved in this process.Receptor protein tyrosine kinases are believed to direct cellular growthvia ligand-stimulated tyrosine phosphorylation of intracellularsubstrates. The class I subfamily of growth factor receptor proteintyrosine kinases includes the 170 kDa epidermal growth factor receptor(EGFR) encoded by the erbB1 gene. erbB1 has been causally implicated inhuman malignancy. In particular, increased expression of this gene hasbeen observed in carcinomas of the breast, bladder, lung, head, neck andstomach. Monoclonal antibodies directed against the EGFR have beenevaluated as therapeutic agents in the treatment of such malignancies.For a review, see Baselga et al. Pharmac. Ther. 64:127-154 (1994). Seealso Masui et al. Cancer Research 44:1002-1007 (1984).

Wu et al. J. Clin. Invest. 95:1897-1905 (1995) recently report that theanti-EGFR monoclonal antibody (mAb) 225 (which competitively inhibitsEGF binding and blocks activation of this receptor) could induce thehuman colorectal carcinoma cell line DiFi (which expresses high levelsof EGFR) to undergo G₁ cell cycle arrest and programmed cell death(apoptosis). Addition of IGF-1 or high concentrations of insulin coulddelay apoptosis induced by mAb 225, whereas G₁ arrest could not bereversed by addition of IGF-1 or insulin.

The second member of the class I subfamily, p185^(neu), was originallyidentified as the product of the transforming gene from neuroblastomasof chemically treated rats. The activated form of the neu protooncogeneresults from a point mutation (valine to glutamic acid) in thetransmembrane region of the encoded protein. Amplification of the humanhomolog of neu (called erbB2 or HER2) is observed in breast and ovariancancers and correlates with a poor prognosis (Slamon et al., Science,235:177-182 (1987); and Slamon et al., Science, 244:707-712 (1989)).Accordingly, Slamon et al. in U.S. Pat. No. 4,968,603 describe variousdiagnostic assays for determining erbB2 gene amplification or expressionin tumor cells. To date, no point mutation analogous to that in the neuprotooncogene has been reported for human tumors. Overexpression(frequently but not uniformly due to amplification) of erbB2 has alsobeen observed in other carcinomas including carcinomas of the stomach,endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas andbladder. See, among others, King et al., Science, 229:974 (1985); Yokotaet al., Lancet. 1:765-767 (1986); Fukushigi et al., Mol Cell Biol.,6:955-958 (1986); Geurin et al., Oncogene Res., 3:21-31 (1988); Cohen etal., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res., 51:1034(1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al.,Cancer Res., 50:421-425 (1990); Kem et al., Cancer Res., 50:5184 (1990);Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog.,3:354-357 (1990); Aasland et al. Br. J. Cancer 57:358-363 (1988);Williams et al. Pathiobiology 59:46-52 (1991); and McCann et al.,Cancer, 65:88-92 (1990).

Antibodies directed against the rat neu and human erbB2 protein productshave been described. Drebin et al., Cell 41:695-706 (1985) refer to anIgG2a monoclonal antibody which is directed against the rat neu geneproduct. This antibody called 7.16.4 causes down-modulation of cellsurface p185 expression on B104-1-1 cells (NIH-3T3 cells transfectedwith the neu protooncogene) and inhibits colony formation of thesecells. Drebin et al. say at page 699 that the antibody exerts acytostatic effect rather than an irreversible cytotoxic effect onneu-transformed cells in soft agar. In Drebin et al. PNAS (USA)83:9129-9133 (1986), the 7.16.4 antibody was shown to inhibit thetumorigenic growth of neu-transformed NIH-3T3 cells as well as ratneuroblastoma cells (from which the neu oncogene was initially isolated)implanted into nude mice. Drebin et al. in Oncogene 2:387-394 (1988)discuss the production of a panel of antibodies against the rat neu geneproduct. All of the antibodies were found to exert a cytostatic effecton the growth of neu-transformed cells suspended in soft agar.Antibodies of the IgM, IgG2a and IgG2b isotypes were able to mediatesignificant in vitro lysis of neu-transformed cells in the presence ofcomplement, whereas none of the antibodies were able to mediate highlevels of antibody-dependent cellular cytotoxicity (ADCC) of theneu-transformed cells. Drebin et al. Oncogene 2:273-277 (1988) reportthat mixtures of antibodies reactive with two distinct regions on thep185 molecule result in synergistic anti-tumor effects onneu-transformed NIH-3T3 cells implanted into nude mice. Biologicaleffects of anti-neu antibodies are reviewed in Myers et al., Meth.Enzym. 198:277-290 (1991). See also WO94/22478 published Oct. 13, 1994.

Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe thegeneration of a panel of anti-ErbB2 antibodies which were characterizedusing the human breast tumor cell line SKBR3. Relative cellproliferation of the SKBR3 cells following exposure to the antibodieswas determined by crystal violet staining of the monolayers after 72hours. Using this assay, maximum inhibition was obtained with theantibody called 4D5 which inhibited cellular proliferation by 56%. Otherantibodies in the panel, including 7C2 and 7F3, reduced cellularproliferation to a lesser extent in this assay. Hudziak et al. concludethat the effect of the 4D5 antibody on SKBR3 cells was cytostatic ratherthan cytotoxic, since SKBR3 cells resumed growth at a nearly normal ratefollowing removal of the antibody from the medium. The antibody 4D5 wasfurther found to sensitize p185^(erB2)-overexpressing breast tumor celllines to the cytotoxic effects of TNF-α. See also WO89/06692 publishedJul. 27, 1989. The anti-ErbB2 antibodies discussed in Hudziak et al. arefurther characterized in Fendly et al. Cancer Research 50:1550-1558(1990); Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. GrowthRegulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol.11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986(1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993);Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. CancerResearch 54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem.269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5(1991); and D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994).

Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe twoantibodies which were selected for their reactivity on the lungadenocarcinoma cell line (Calu-3) which overexpresses ErbB2. One of theantibodies, called MGR3, was found to internalize, inducephosphorylation of ErbB2, and inhibit tumor cell growth in vitro.

McKenzie et al. Oncogene 4:543-548 (1989) generated a panel ofanti-ErbB2 antibodies with varying epitope specificities, including theantibody designated TA1. This TA1 antibody was found to induceaccelerated endocytosis of ErbB2 (see Maier et al. Cancer Res.51:5361-5369 (1991)). Bacus et al. Molecular Carcinogenesis 3:350-362(1990) reported that the TA1 antibody induced maturation of the breastcancer cell lines AU-565 (which overexpresses the erbB2 gene) and MCF-7(which does not). Inhibition of growth and acquisition of a maturephenotype in these cells was found to be associated with reduced levelsof ErbB2 receptor at the cell surface and transient increased levels inthe cytoplasm.

Stancovski et al. PNAS (USA) 88:8691-8695 (1991) generated a panel ofanti-ErbB2 antibodies, injected them i.p. into nude mice and evaluatedtheir effect on tumor growth of murine fibroblasts transformed byoverexpression of the erbB2 gene. Various levels of tumor inhibitionwere detected for four of the antibodies, but one of the antibodies(N28) consistently stimulated tumor growth. Monoclonal antibody N28induced significant phosphorylation of the ErbB2 receptor, whereas theother four antibodies generally displayed low or nophosphorylation-inducing activity. The effect of the anti-ErbB2antibodies on proliferation of SKBR3 cells was also assessed. In thisSKBR3 cell proliferation assay, two of the antibodies (N12 and N29)caused a reduction in cell proliferation relative to control. Theability of the various antibodies to induce cell lysis in vitro viacomplement-dependent cytotoxicity (CDC) and antibody-mediatedcell-dependent cytotoxicity (ADCC) was assessed, with the authors ofthis paper concluding that the inhibitory function of the antibodies wasnot attributed significantly to CDC or ADCC.

Bacus et al. Cancer Research 52:2580-2589 (1992) further characterizedthe antibodies described in Bacus et al. (1990) and Stancovski et al. ofthe preceding paragraphs. Extending the i.p. studies of Stancovski etal., the effect of the antibodies after i.v. injection into nude miceharboring mouse fibroblasts overexpressing human ErbB2 was assessed. Asobserved in their earlier work, N28 accelerated tumor growth whereas N12and N29 significantly inhibited growth of the ErbB2-expressing cells.Partial tumor inhibition was also observed with the N24 antibody. Bacuset al. also tested the ability of the antibodies to promote a maturephenotype in the human breast cancer cell lines AU-565 and MDA-MB453(which overexpress ErbB2) as well as MCF-7 (containing low levels of thereceptor). Bacus et al. saw a correlation between tumor inhibition invivo and cellular differentiation; the tumor-stimulatory antibody N28had no effect on differentiation, and the tumor inhibitory action of theN12, N29 and N24 antibodies correlated with the extent ofdifferentiation they induced.

Xu et al. Int. J. Cancer 53:401-408 (1993) evaluated a panel ofanti-ErbB2 antibodies for their epitope binding specificities, as wellas their ability to inhibit anchorage-independent andanchorage-dependent growth of SKBR3 cells (by individual antibodies andin combinations), modulate cell-surface ErbB2, and inhibit ligandstimulated anchorage-independent growth. See also WO94/00136 publishedJan. 6, 1994 and Kasprzyk et al. Cancer Research 52:2771-2776 (1992)concerning anti-ErbB2 antibody combinations. Other anti-ErbB2 antibodiesare discussed in Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawveret al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. Cancer Res.54:3758-3765 (1994); and Harwerth et al. J. Biol. Chem. 267:15160-15167(1992).

A further gene related to erbB2, called erbB3 or HER3, has also beendescribed. See, e.g., U.S. Pat. Nos. 5,183,884 and 5,480,968. ErbB3 isunique among the ErbB receptor family in that it possesses little or nointrinsic tyrosine kinase activity. However, when ErbB3 is co-expressedwith ErbB2, an active signaling complex is formed and antibodiesdirected against ErbB2 are capable of disrupting this complex(Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994)).Additionally, the affinity of ErbB3 for heregulin (HRG) is increased toa higher affinity state when co-expressed with ErbB2. See also, Levi etal., Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al.,Proc. Natl. Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al.,Cancer Res., 56:1457-1465 (1996) with respect to the ErbB2-ErbB3 proteincomplex.

The class I subfamily of growth factor receptor protein tyrosine kinaseshas been further extended to include the HER4/p180^(erB4) receptor. SeeEP Pat Appln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA,90:1746-1750 (1993); and Plowman et al., Nature, 366:473-475 (1993).Plowman et al. found that increased HER4 expression correlated withcertain carcinomas of epithelial origin, including breastadenocarcinomas. This receptor, like ErbB3, forms an active signallingcomplex with ErbB2 (Carraway and Cantley, Cell 78:5-8 (1994)).

The quest for an ErbB2 activator has lead to the discovery of a familyof heregulin polypeptides. These proteins appear to result fromalternative splicing of a single gene and are called neuregulins (NRGs),neu differentiation factors (NDFs), heregulins (HRGs), glial growthfactors (GGFs) and acetylcholine receptor inducing activity (ARIA) inthe literature. For a review, see Groenen et al. Growth Factors11:235-257 (1994); Lemke, G. Molec. & Cell. Neurosci. 7:247-262 (1996)and Lee et al. Pharm. Rev. 47:51-85 (1995).

SUMMARY OF THE INVENTION

This invention relates, at least in part, to the surprising discoverythat certain anti-ErbB2 antibodies can induce death of an ErbB2overexpressing cell (e.g. a BT474, SKBR3, SKOV3 or Calu 3 cell) viaapoptosis. In contrast to the apoptotic anti-EGFR antibody described inWu et al., J. Clin. Invest. 95:1897-1905 (1995), the anti-ErbB2antibodies of interest herein are not thought to induce apoptosis bydisruption of an autocrine loop. Antibodies herein with these celldeath-inducing attributes will normally bind to a region in theextracellular domain of ErbB2, e.g. to an epitope in Domain 1 of ErbB2.Preferably, the antibodies will bind to the ErbB2 epitope bound by the7C2 and/or 7F3 antibodies described herein.

Preferred antibodies are monoclonal antibodies e.g. humanizedantibodies. Antibodies of particular interest are those which, inaddition to the above-described properties, bind the ErbB2 receptor withan affinity of at least about 10 nM, more preferably at least about 1nM.

In certain embodiments, the antibody is immobilized on (e.g. covalentlyattached to) a solid phase, e.g. for affinity purification of thereceptor or for diagnostic assays. For diagnostic uses, it may also bebeneficial to provide a labelled antibody (i.e. the antibody bound to adetectable label).

The antibodies of the preceding paragraphs may be provided in the formof a composition comprising the antibody and a pharmaceuticallyacceptable carrier or diluent. Optionally, the composition furthercomprises a second anti-ErbB2 antibody, especially where the secondanti-ErbB2 antibody is one which binds to an epitope on the ErbB2receptor which differs from that to which the 7C2/7F3 antibodiesdisclosed herein bind. In a preferred embodiment, the second antibody isone which inhibits growth of SKBR3 cells in cell culture by 50%-100%(i.e. 4D5 antibody and functional equivalents thereof). The compositionfor therapeutic use will be sterile and may be lyophilized.

The invention also provides: an isolated nucleic acid molecule encodingthe antibody of the preceding paragraphs which may further comprise apromoter operably linked thereto; an expression vector comprising thenucleic acid molecule operably linked to control sequences recognized bya host cell transformed with the vector; a host cell comprising thenucleic acid (e.g. a hybridoma cell line); and a process for making theantibody comprising culturing a cell comprising the nucleic acid so asto express the anti-ErbB2 antibody and, optionally, recovering theantibody from the host cell culture and, preferably, the host cellculture medium.

The invention also provides methods for using the anti-ErbB2 antibodiesdisclosed herein. For example, the invention provides a method forinducing cell death comprising exposing a cell, such as a cancer cellwhich overexpresses ErbB2, to anti-ErbB2 antibody described herein in anamount effective to induce cell death. The cell may be in cell cultureor in a mammal, e.g. a mammal suffering from cancer. The invention alsoprovides a method for inducing apoptosis of a cell which overexpressesErbB2 comprising exposing the cell to exogenous anti-ErbB2 antibody asdescribed herein in an amount effective to induce apoptosis of the cell.Thus, the invention provides a method for treating a mammal sufferingfrom a condition characterized by overexpression of the ErbB2 receptor,comprising administering a pharmaceutically effective amount of theanti-ErbB2 antibodies disclosed herein to the mammal. According to anyof the above methods, a further anti-ErbB2 antibody may be used,especially one which binds to a different ErbB2 epitope from that towhich the 7C2/7F3 antibodies disclosed herein bind (e.g. one that doesnot bind Domain 1). In one embodiment, the second antibody inhibitsgrowth of SKBR3 cells in cell culture by 50%-100% and optionally bindsto the epitope on ErbB2 to which 4D5 binds.

In Example 2 below, it was found that the pro-apoptotic antibody 7C2almost completely eradicated the entire culture of growth arrestedcells. Therefore, it may be desirable to combine the pro-apoptoticantibodies disclosed herein with a growth inhibitory agent in the invitro and in vivo methods discussed above. In such embodiments, superiorlevels of apoptosis may be achieved by administering the growthinhibitory agent prior to the pro-apoptotic anti-ErbB2 antibody.However, simultaneous administration or administration of the anti-ErbB2antibody first is also contemplated.

The invention also provides an article of manufacture for use in theabove in vivo methods which comprises a container holding the anti-ErbB2antibody and a label on or associated with the container which indicatesthat the antibody can be used to treat conditions characterized by ErbB2overexpression, such as cancer.

In a further aspect, the invention provides a method for detecting ErbB2in vitro or in vivo comprising contacting the antibody with a cellsuspected of containing ErbB2 and detecting if binding has occurred.Accordingly, the invention provides an assay for detecting a tumorcharacterized by amplified expression of ErbB2 comprising the steps ofexposing a cell to the antibody disclosed herein and determining theextent of binding of the antibody to the cell. Preferably the antibodyfor use in such an assay will be labelled and will be supplied in theform of a kit with instructions for using the antibody to detect ErbB2.The assay herein may be an in vitro assay (such as an ELISA assay) or anin vivo assay. For in vivo tumor diagnosis, the antibody is preferablyconjugated to a radioactive isotope and administered to a mammal, andthe extent of binding of the antibody to tissues in the mammal isobserved by external scanning for radioactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & B show the effects of apoptosis on a cell and a method fordetermining apoptosis, respectively. FIG. 1A shows the physiologicalchanges which occur to a cell which undergoes programmed cell death(apoptosis). FIG. 1B shows translocation of phosphatidyl serine (PS)from the inner leaflet of the plasma membrane to the exterior of thecell. This PS translocation process is specific for cells undergoingapoptosis. Annexin V binds specifically to PS and thus provides a meansfor determining apoptosis.

FIG. 2 shows the epitope specificity of anti-ErbB2 antibodies. MAbs wereused to block the binding of 7C2 to BT474 cells. BT474 cells (0.5×10⁶)pretreated or not with 50 μg anti-ErbB2 antibodies (15 minutes on ice)were washed twice, resuspended in 0.1 ml 1% FBS/PBS and incubated with 5μg of fluoresceinisothiocyanate (FITC) conjugated 7C2 antibody (15minutes on ice). Following incubation, the cell suspensions were washedtwice with 1% FBS/PBS to remove unbound fluorochrome, fixed with 1%paraformaldehyde/PBS and analyzed by flow cytometry.

FIGS. 3A-D depict the effect of anti-ErbB2 antibodies on human breastcancer cells which overexpress ErbB2. Cells with normal membranefunctions at the time of harvest (“viable” cells) exclude the DNA dye7AAD and after membrane permeabilization are stained preferentially withHoechst. Analysis of these cells reveals a classic DNA profile (farright panels) with cells in the G₀/G₁ phase (major peak) and S-G₂-Mphase (indicated with double headed arrow) of the cell cycle. In thecenter panels, the population of cells indicated by the arrow representdead cells, some of which had abnormal permeability characteristics atthe time of harvest and had degraded their nuclear DNA. 4×10⁴ BT474cells/ml were incubated for 72 hours in medium containing anisotype-matched control Ig (FIG. 3A), 1 μg of monoclonal 4D5 (FIG. 3B),50 μg of monoclonal 7C2 (FIG. 3C), or 1 μg 4D5 antibody+50 μg 7C2antibody (FIG. 3D). The percentage of dead cells is indicated in themiddle panels (7AAD fluorescence vs. Hoechst fluorescence) and thepercentage of viable cells in the combined S, G₂, and M phases of thecell cycle is indicated in the right panels (Hoechst fluorescence vs.cell count). The lefthand panel shows the size of the cells asdetermined by forward and right angle light scatter.

FIGS. 4A & B reveal the additive effect of 4D5 and 7C2 antibodies on DNAsynthesis, and viability in BT474 cells. The average of twoexperiments±S.D. is shown. In FIG. 4A, 8×10³ cells/0.2 ml/well weretreated with 4D5 (0.05 μg/ml) and/or 7C2 (50 μg/ml) for 72 hours andpulsed for the last 12 hours with 1 μCi [³H]-thymidine (in triplicate).In FIG. 4B, for cell viability, a total cell count was obtained andviability was determined by FACS analysis. The standard deviation forboth 7C2 and 7C2 plus 4D5 treatment is too small (1×10³ cells) to beseen in the figure.

FIGS. 5A-F show induction of apoptosis by anti-ErbB2 MAbs 7C2 and 4D5 inBT474 breast tumor cells. FIGS. 5A, 5C and 5E show plots of forwardscatter (FS), an indicator of cell size, vs. log FITC (representingannexin V binding). FIGS. 5B, D and F are quadrant plots of log FITC vs.log propidium iodine (PI), with percent annexin V-positive cells shownin quadrant 4 and percent annexin V/PI positive cells in quadrant 2.Untreated cells display a uniform size and fluorescence signal, as seenwithin the drawn circle, and are 85% viable (FIG. 5A). In addition todisplaying low annexin V-binding, these cells do not take up PI,indicating no change in membrane integrity. Treatment for 3 days with 10μg/ml MAb 7C2 results in a reduction in the percent of viable cells (to25.3%, FIG. 5E) and a shift of almost the entire population to asmaller, FITC-positive population of cells (FIG. 5E). As shown in FIG.5F, MAb 7C2 induces a 7-8 fold increase in the percent of annexinV-positive/PI-positive cells, indicating apoptotic cell death. Theanti-proliferative MAb, 4D5, induces a small degree of apoptosis (2.5fold above control, FIGS. 5C and D).

FIG. 6 shows that the effects of MAb 7C2 are dose-dependent. Theinduction of apoptosis in BT474 breast tumor cells by MAb 7C2, asmeasured by an increase in the number of annexin V-positive andPI-positive cells, is apparent at a concentration of 0.1 μg/ml andreaches a maximum at 1 μg/ml.

FIGS. 7A and 7B are time-courses of MAb 7C2-induced apoptosis in BT474and SKBR3 breast tumor cells, respectively. Treatment of BT474 cells(FIG. 7A) and SKBR3 cells (FIG. 78) with 10 μg/ml MAb 7C2 results in areduction in the percent of viable cells (those cells which are annexinV- and PI-negative) as early as 15 minutes after initiation of treatmentand reaches a maximum at 24 hours. The BT474 cell line is more sensitiveto the pro-apoptotic effect of MAb 7C2 compared to the SKBR3 cells.

FIGS. 8A-E show responses of different cell lines to anti-ErbB2 MAbs.The BT474, SKBR3 and MCF7 breast tumor cell lines, and normal humanmammary epithelial cells (HMEC) (FIGS. 8A-D, respectively) wereincubated with the anti-ErbB2 MAbs 4D5, 3H4, 7F3, 7C2, 2H11, 3E8, and7D3; the humanized version of muMAb 4D5 (hu4D5); or the isotype-matchedirrelevant control MAb 1766. Treatment was for 3 days at a MAbconcentration of 10 μg/ml. The data are pooled from 2-9 separateexperiments and are represented as mean fold increase (+/−s.e.) inannexin V binding over control cells. The response of the BT474 breasttumor cells to MAbs 7C2 and 4D5 is as described for FIG. 5 (9-fold and2.5-fold above control, respectively). Induction of apoptosis in theSKBR3 breast tumor cell line, which expresses high levels of ErbB2similar to the BT474 cells, also occurs after treatment with MAb 7C2(and to a smaller degree, MAb 4D5). In addition, MAb 7F3 inducesapoptosis in both the BT474 and SKBR3 cell lines. The MCF7 breast tumorline, which expresses normal ErbB2 levels, and the HMEC's showed nochange in annexin V binding after treatment with the anti-ErbB2 Mabs.These results suggest that overexpression of ErbB2 is required forresponsiveness to the anti-ErbB2 MAbs. In FIG. 8E, a non-small cell lungcarcinoma line overexpressing ErbB2, Calu 3, was tested for induction ofapoptosis by anti-ErbB2 MAbs. Treatment with 7C2 or 7F3 resulted inenhanced binding of annexin V.

FIGS. 9A-I show the effects of MAbs 7C2 and 4D5 on cell cycleprogression and cell death. Untreated BT474 cells are largely annexinV-negative and PI-negative (FIG. 9A, quadrant 3), and show a normal cellcycle DNA histogram (FIGS. 9B &C). Cells treated with 10 μg/ml MAb 4D5show some increase in uptake of PI and annexin V-FITC binding (FIG. 9D,quadrant 2). The most pronounced effect is on cell cycle progression,where MAb 4D5 almost completely reduces the percent of cells in S phase(FIGS. 9E &F). MAb 7C2 induces a significant amount of cell death inBT474, as measured by PI uptake and annexin V-FITC binding (FIG. 9G,quadrant 2). Cell cycle analysis shows the presence of a sub-G₀/G₁ orhypodiploid population (FIG. 9I), characteristic of apoptotic cells,with the cells displaying high levels of annexin V binding (FIG. 9H,quadrant 1).

FIGS. 10A-F are the results from curve-fitting analyses of the DNAhistograms of FIGS. 9A-I and show little change in the percent of cellsin S-phase (52%) after MAb 7C2 treatment compared to control cells (61%S-phase cells), but a dramatic reduction in the number of cells inS-phase (to 6%) in response to MAb 4D5 (FIGS. 10C, A and B,respectively). Analyses of the apoptotic population of cells (annexinV/PI positive cells from quadrant 2, 9A, D and G) reveal no differencein the percent S-phase cells compared to the total cell population (FIG.10D control=55%; FIG. 10E MAb 4D5=7%; FIG. 10F MAb 7C2=56%).Furthermore, the G_(d)/G₁ and G₂/M phases show no change (compare FIGS.10A and D, B and E, C and F), indicating that cells are exiting the cellcycle at all phases and that MAb-induced apoptotic cell death is notcell cycle specific.

FIGS. 11A & B show MAb 7C2-induced apoptosis is enhanced by growtharrest. In addition to the data from cell cycle studies, it wasobserved, from video time-lapse recording, that a proportion of MAb7C2-treated cells continue to proliferate while others undergoapoptosis. Therefore, experiments were performed to determine ifinhibition of cell growth would enhance the pro-apoptotic activity ofMAb 7C2. BT474 cells were serum-deprived for 3 days to induce growtharrest, then treated with 10 μg/ml MAb 7C2 or 4D5 for 3 days andanalyzed for viability (annexin V-FITC binding and PI uptake) as well ascell cycle effects. Serum-deprivation (by incubation in mediasupplemented with 0.1% FBS) effectively reduces proliferation, as seenby a decrease in the percent of S-phase cells from 33% (in 10% FBS) to10% (FIG. 11A). The potent anti-proliferative activity of MAb 4D5 is notfurther enhanced by prior growth arrest. The proportion of cells inS-phase, with and without serum-deprivation, in MAb 7C2-treated cellswas similar to controls. FIG. 11B shows that serum-starvation does notadversely affect cell viability of untreated cells. However, viabilityis reduced to 55% in BT474 cells treated with 10 μg/ml MAb 4D5 after aperiod of serum-deprivation. Moreover, treatment of growth-arrestedcells with 10 μg/ml MAb 7C2 almost completely eradicates the entireculture, in that the percent of annexin V-negative and PI-negative cellsis reduced to 10%.

FIG. 12 depicts with underlining the amino acid sequence of Domain 1 ofErbB2 (SEQ ID NO:1). Bold amino acids indicate the location of theepitope recognized by MAbs 7C2 and 7F3 as determined by deletionmapping, i.e. the “7C2/7F3 epitope” (SEQ ID NO:2).

FIG. 13 shows epitope-mapping of the extracellular domain of ErbB2 asdetermined by truncation mutant analysis and site-directed mutagenesis(Nakamura et al. J. of Virology 67(10):6179-6191 (October 1993); Renz etal. J. Cell Biol. 125(6):1395-1406 (June 1994)). Pro-apoptotic MAbs 7C2and 7F3 bind an epitope at the N-terminus of the receptor, whereasanti-proliferative MAbs 4D5 and 3H4 bind adjacent to the transmembranedomain. The various ErbB2-ECD truncations or point mutations wereprepared from cDNA using polymerase chain reaction technology. The ErbB2mutants were expressed as gD fusion proteins in a mammalian expressionplasmid. This expression plasmid uses the cytomegaloviruspromoter/enhancer with SV40 termination and polyadenylation signalslocated downstream of the inserted cDNA. Plasmid DNA was transfectedinto 293S cells. One day following transfection, the cells weremetabolically labeled overnight in methionine and cysteine-free, lowglucose DMEM containing 1% dialyzed fetal bovine serum and 25 μCi eachof ³⁵S methionine and ⁵S cysteine. Supernatants were harvested eitherthe ErbB2 MAbs or control antibodies were added to the supernatant andincubated 2-4 hours at 4° C. The complexes were precipitated, applied toa 10-20% Tricine SDS gradient gel and electrophoresed at 100 V. The gelwas electroblotted onto a membrane and analyzed by autoradiography.

FIGS. 14A-E show that the effects of anti-ErbB2 MAbs areepitope-specific. To determine if the anti-proliferative orpro-apoptotic effects of anti-ErbB2 MAbs are related to epitopespecificity, BT474 cells were treated with 4 different MAbs for 3 daysand stained with Hoechst 33342 for cell cycle analysis. The MAbs were:7C2 and 7F3, which bind amino acids 22-53 (SEQ ID NO:2) on the ErbB2extracellular domain (FIGS. 14B and C, respectively); 4D5, which bindsresidues 529-625 (SEQ ID NO:4) (FIG. 14D); and 3H4, which binds aminoacids 541-599 (SEQ ID NO:3) (FIG. 14E). Both 7C2 and 7F3 induceapoptosis (to 60.5% and 53.4%, respectively, of the cell population),but did not decrease the proportion of S-phase cells (64.9% and 58.7%,respectively) compared to untreated cells (52.5%; FIG. 14A). Incontrast, MAbs 4D5 and 3H4, which bind adjacent to the ErbB2transmembrane region, show potent anti-proliferative activity (% S=5.4and 10.5, respectively, control S=52.5%), but are not as effective as7C2 or 7F3 in promoting apoptotic cell death (% apoptosis for 4D5=41.9,for 3H4=26.3, controls=15.8%).

FIG. 15 shows induction of apoptosis by anti-HER2MAb 7C2 in the SKOV3ovarian carcinoma cell line as determined in Example 3.

FIG. 16 shows that combination treatment with anti-HER2MAbs results inenhanced apoptotic effects on BT474 breast tumor cells whereanti-HER2MAb 7C2 is administered prior to anti-HER2MAb 4D5 (see Example3).

FIG. 17 shows the effects of administration of anti-HER2MAbs alone or incombination on mean tumor volume (mm³)+/−1 S.E. of BT474M1 xenografts innude mice as described in Example 4. Antibodies were administered twiceweekly beginning on day 6.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

Unless indicated otherwise, the term “ErbB2” when used herein refers tohuman ErbB2 protein and “erbB2” refers to human erbB2 gene. The humanerbB2 gene and ErbB2 protein are described in Semba et al., PNAS (USA)82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234 (1986)(Genebank accession number X03363), for example. ErbB2 comprises fourdomains (Domains 1-4). “Domain 1” at the amino terminus of theextracellular domain of ErbB2 is shown in FIG. 12 herein. See Plowman etal. Proc. Natl. Acad. Sci. USA 90:1746-1750 (1993).

The “epitope 7C2/7F3” is the region at the N terminus of theextracellular domain of ErbB2 to which the 7C2 and/or 7F3 antibodies(each deposited with the ATCC, see below) bind. To screen for antibodieswhich bind to the 7C2/7F3 epitope, a routine cross-blocking assay suchas that described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed (see Example 2 below) toestablish whether the antibody binds to the 7C2/7F3 epitope on ErbB2(i.e. any one or more of residues in the region from about residue 22 toabout residue 53 of ErbB2 (SEQ ID NO:2)).

The “epitope 4D5” is the region in the extracellular domain of ErbB2 towhich the antibody 4D5 (ATCC CRL 10463) binds. This epitope is close tothe transmembrane region of ErbB2. To screen for antibodies which bindto the 4D5 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed (see Example 2 below) toassess whether the antibody binds to the 4D5 epitope of ErbB2 (i.e. anyone or more residues in the region from about residue 529, e.g. aboutresidue 561 to about residue 625, inclusive (SEQ ID NO:4)).

The term “induces cell death” refers to the ability of the antibody tomake a viable cell become nonviable. The “cell” here is one whichexpresses the ErbB2 receptor, especially where the cell overexpressesthe ErbB2 receptor. A cell which “overexpresses” ErbB2 has significantlyhigher than normal ErbB2 levels compared to a noncancerous cell of thesame tissue type.

Preferably, the cell is a cancer cell, e.g. a breast, ovarian, stomach,endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic orbladder cell. In vitro, the cell may be a SKBR3, BT474, Calu 3,MDA-MB-453, MDA-MB-361 or SKOV3 cell. Cell death in vitro may bedetermined in the absence of complement and immune effector cells todistinguish cell death induced by antibody dependent cellularcytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus,the assay for cell death may be performed using heat inactivated serum(i.e. in the absence of complement) and in the absence of immuneeffector cells. To determine whether the antibody is able to induce celldeath, loss of membrane integrity as evaluated by uptake of propidiumiodide (PI) (see Example 2 below), trypan blue (see Moore et al.Cytotechnology 17:1-11 (1995)) or 7AAD (see Example 1 below) can beassessed relative to untreated cells. Preferred cell death-inducingantibodies are those which induce PI uptake in the “PI uptake assay inBT474 cells” (see below)

The phrase “induces apoptosis” refers to the ability of the antibody toinduce programmed cell death as determined by binding of annexin V,fragmentation of DNA, cell shrinkage, dilation of endoplasmaticreticulum, cell fragmentation, and/or formation of membrane vesicles(called apoptotic bodies). See FIGS. 1A & B herein. The cell is onewhich overexpresses the ErbB2 receptor. Preferably the “cell” is a tumorcell, e.g. a breast, ovarian, stomach, endometrial, salivary gland,lung, kidney, colon, thyroid, pancreatic or bladder cell. In vitro, thecell may be a SKBR3, BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or SKOV3cell. Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding (see Example 2 below);DNA fragmentation can be evaluated through DNA laddering as disclosed inExample 2 herein; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody which induces apoptosis is one which results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcell in an “annexin binding assay using BT474 cells” (see below).

Sometimes the pro-apoptotic antibody will be one which blocks HRGbinding/activation of the ErbB2/ErbB3 complex (e.g. 7F3 antibody). Inother situations, the antibody is one which does not significantly blockactivation of the ErbB2/ErbB3 receptor complex by HRG (e.g. 7C2).Further, the antibody may be one like 7C2 which, while inducingapoptosis, does not induce a large reduction in the percent of cells inS phase (e.g. one which only induces about 0-10% reduction in thepercent of these cells relative to control as determined in FIG. 10).

The antibody of interest may be one like 7C2 which binds specifically tohuman ErbB2 and does not significantly cross-react with other proteinssuch as those encoded by the erbB1, erbB3 and/or erbB4 genes. Sometimes,the antibody may not significantly cross-react with the rat neu protein,e.g., as described in Schecter et al. Nature 312:513 (1984) and Drebinet al., Nature 312:545-548 (1984). In such embodiments, the extent ofbinding of the antibody to these proteins (e.g., cell surface binding toendogenous receptor) will be less than about 10% as determined byfluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA).

“Heregulin” (HRG) when used herein refers to a polypeptide whichactivates the ErbB2-ErbB3 and ErbB2-ErbB4 protein complexes (i.e.induces phosphorylation of tyrosine residues in the complex upon bindingthereto). Various heregulin polypeptides encompassed by this term aredisclosed in Holmes et al., Science, 256:1205-1210 (1992); WO 92/20798;Wen et al., Mol. Cell. Biol., 14(3):1909-1919 (1994); and Marchionni etal., Nature, 362:312-318 (1993), for example. The term includesbiologically active fragments and/or variants of a naturally occurringHRG polypeptide, such as an EGF-like domain fragment thereof (e.g.HRGβ1₁₇₇₋₂₄₄).

The “ErbB2-ErbB3 protein complex” and “ErbB2-ErbB4 protein complex” arenoncovalently associated oligomers of the ErbB2 receptor and the ErbB3receptor or ErbB4 receptor, respectively. The complexes form when a cellexpressing both of these receptors is exposed to HRG and can be isolatedby immunoprecipitation and analyzed by SDS-PAGE as described inSliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994).

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., NIH Publ. No.91-3242, Vol. I, pages 647-669 (1991)). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (K) and lambda (A), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antibody” is used in the broadest sense and specificallycovers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al. Protein Eng.8(10):1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanizedantibody includes a PRIMATIZED™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V) in thesame polypeptide chain (V_(H)-V_(L). By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

A “disorder” is any condition that would benefit from treatment with theanti-ErbB2 antibody. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include benign and malignant tumors; leukemias andlymphoid malignancies; neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeAdriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (“Ara-C”),Cyclophosphamide, Thiotepa; Busulfan, Cytoxin, Taxol, Toxotere,Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide,Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine,Carboplatin, Teniposide, Daunomycin, Caminomycin, Aminopterin,Dactinomycin, Mitomycins, Esperamicins (see U.S. Pat. No. 4,675,187),Melphalan and other related nitrogen mustards. Also included in thisdefinition are hormonal agents that act to regulate or inhibit hormoneaction on tumors such as tamoxifen and onapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially anErbB2-overexpressing cancer cell either in vitro or in vivo. Thus, thegrowth inhibitory agent is one which significantly reduces thepercentage of ErbB2 overexpressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce G1 arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), taxol, and topo II inhibitors such asdoxorubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogens, and antineoplastic drugs by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The 4D5 antibody (and functionalequivalents thereof) can also be employed for this purpose.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labelled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the anti-ErbB2 antibodies disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Preferably, “operablylinked” means that the DNA sequences being linked are contiguous, and,in the case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

II. Modes for Carrying out the Invention

A. Antibody Preparation

A description follows as to exemplary techniques for the production ofthe claimed antibodies. The ErbB2 antigen to be used for production ofantibodies may be, e.g., a soluble form of the extracellular domain ofErbB2; a peptide such as a Domain 1 peptide or a portion thereof (e.g.comprising the 7C2 or 7F3 epitope). Alternatively, cells expressingErbB2 at their cell surface (e.g. NIH-3T3 cells transformed tooverexpress ErbB2, see Examples 1 & 2 below; or a carcinoma cell linesuch as SKBR3 cells, see Stancovski et al. PNAS (USA) 88:8691-8695(1991)) can be used to generate antibodies. Other forms of ErbB2 usefulfor generating antibodies will be apparent to those skilled in the art.

(i) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites.

One month later the animals are boosted with 1/5 to 1/10 the originalamount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized and Human Antibodies

Methods for humanizing non-human antibodies are well known in the art.Preferably, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).

(iv) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185.

(v) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the ErbB2 protein. For example, onearm may bind an epitope in Domain 1 of ErbB2 such as the 7C2/7F3epitope, the other may bind a different ErbB2 epitope, e.g. the 4D5epitope. Other such antibodies may combine an ErbB2 binding site withbinding site(s) for EGFR, ErbB3 and/or ErbB4. Alternatively, ananti-ErbB2 arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 orCD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII(CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms tothe ErbB2-expressing cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express ErbB2. These antibodiespossess an ErbB2-binding arm and an arm which binds the cytotoxic agent(e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain,methotrexate or radioactive isotope hapten). Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the C_(H)3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

(vi) Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. Thoseantibodies having the characteristics described herein are selected.

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., PI, trypan blue or 7AAD uptake isassessed relative to control. The preferred assay is the “PI uptakeassay using BT474 cells”. According to this assay, BT474 cells (whichcan be obtained from the American Type Culture Collection (Rockville,Md.)) are cultured in Dulbecco's Modified Eagle Medium (D-MEM):Ham'sF-12 (50:50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2mM L-glutamine. (Thus, the assay is performed in the absence ofcomplement and immune effector cells). The BT474 cells are seeded at adensity of 3×10⁶ per dish in 100×20 mm dishes and allowed to attachovernight. The medium is then removed and replaced with fresh mediumalone or medium containing 10 μg/ml of the appropriate MAb. The cellsare incubated for a 3 day time period. Following each treatment,monolayers are washed with PBS and detached by trypsinization. Cells arethen centrifuged at 1200 rpm for 5 minutes at 4° C., the pelletresuspended in 3 ml ice cold Ca²⁺ binding buffer (10 mM Hepes, pH 7.4,140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake areselected.

In order to select for antibodies which induce apoptosis, an “annexinbinding assay using BT474 cells” as described in Example 2 below isavailable. The BT474 cells are cultured and seeded in dishes asdiscussed in the preceding paragraph. The medium is then removed andreplaced with fresh medium alone or medium containing 10 μg/ml of theMAb. Following a three day incubation period, monolayers are washed withPBS and detached by trypsinization. Cells are then centrifuged,resuspended in Ca²⁺ binding buffer and aliquoted into tubes as discussedabove for the cell death assay. Tubes then receive labelled annexin(e.g. annexin V-FTIC) (1 μg/ml). Samples may be analyzed using aFACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (BectonDickinson). Those antibodies which induce statistically significantlevels of annexin binding relative to control are selected asapoptosis-inducing antibodies.

In addition to the annexin binding assay discussed in the precedingparagraph, a “DNA staining assay using BT474 cells” is available. Inorder to perform this assay, BT474 cells which have been treated withthe antibody of interest as described in the preceding two paragraphsare incubated with 9 μg/ml HOECHST 33342™ for 2 hr at 37° C., thenanalyzed on an EPICS ELITE™ flow cytometer (Coulter Corporation) usingMODFIT LT™ software (Verity Software House). Antibodies which induce achange in the percentage of apoptotic cells which is 2 fold or greater(and preferably 3 fold or greater) than untreated cells (up to 100%apoptotic cells) may be selected as pro-apoptotic antibodies using thisassay.

To screen for antibodies which bind to an epitope on ErbB2 bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping described in Example 2 can be performed.

To identify anti-ErbB2 antibodies which inhibit growth of SKBR3 cells incell culture by 50-100%, the SKBR3 assay described in WO89/06692 can beperformed. According to this assay, SKBR3 cells are grown in a 1:1mixture of F12 and DMEM medium supplemented with 10% fetal bovine serum,glutamine and penicillinstreptomycin. The SKBR3 cells are plated at20,000 cells in a 35 mm cell culture dish (2 mls/35 mm dish). 2.5 μg/mlof the anti-ErbB2 antibody is added per dish. After six days, the numberof cells, compared to untreated cells are counted using an electronicCOULTER″ cell counter. Those antibodies which inhibit growth of theSKBR3 cells by 50-100% are selected for combination with the apoptoticantibodies as desired.

(vii) Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of the antibodyin treating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992)and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodieswith enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch 53:2560-2565 (1993). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989);

(viii) Immunoconjugates

The invention also pertains to immunoconjugates comprising the antibodydescribed herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated anti-ErbB2 antibodies. Examples include ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

(ix) Immunoliposomes

The anti-ErbB2 antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al. J. National Cancer Inst.81(19)1484 (1989)

(x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibody of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as (3-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs;(3-lactamase useful for converting drugs derivatized with β-lactams intofree drugs; and penicillin amidases, such as penicillin V amidase orpenicillin G amidase, useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-ErbB2antibodies by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984)).

(xi) Antibody-Salvage Receptor Binding Epitope Fusions.

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half life. This maybe achieved, for example, by incorporation of a salvage receptor bindingepitope into the antibody fragment (e.g. by mutation of the appropriateregion in the antibody fragment or by incorporating the epitope into apeptide tag that is then fused to the antibody fragment at either end orin the middle, e.g., by DNA or peptide synthesis).

A systematic method for preparing such an antibody variant having anincreased in vivo half-life comprises several steps. The first involvesidentifying the sequence and conformation of a salvage receptor bindingepitope of an Fc region of an IgG molecule. Once this epitope isidentified, the sequence of the antibody of interest is modified toinclude the sequence and conformation of the identified binding epitope.After the sequence is mutated, the antibody variant is tested to see ifit has a longer in vivo half-life than that of the original antibody. Ifthe antibody variant does not have a longer in vivo half-life upontesting, its sequence is further altered to include the sequence andconformation of the identified binding epitope. The altered antibody istested for longer in vivo half-life, and this process is continued untila molecule is obtained that exhibits a longer in vivo half-life.

The salvage receptor binding epitope being thus incorporated into theantibody of interest is any suitable such epitope as defined above, andits nature will depend, e.g., on the type of antibody being modified.The transfer is made such that the antibody of interest still possessesthe biological activities described herein.

The epitope preferably constitutes a region wherein any one or moreamino acid residues from one or two loops of a Fc domain are transferredto an analogous position of the antibody fragment. Even more preferably,three or more residues from one or two loops of the Fc domain aretransferred. Still more preferred, the epitope is taken from the CH2domain of the Fc region (e.g., of an IgG) and transferred to the CH1,CH3, or V_(H) region, or more than one such region, of the antibody.Alternatively, the epitope is taken from the CH2 domain of the Fc regionand transferred to the C_(L) region or V_(L) region, or both, of theantibody fragment.

In one most preferred embodiment, the salvage receptor binding epitopecomprises the sequence (5′ to 3′): PKNSSMISNTP (SEQ ID NO:5), andoptionally further comprises a sequence selected from the groupconsisting of HQSLGTQ (SEQ ID NO:6), HQNLSDGK (SEQ ID NO:7), HQNISDGK(SEQ ID NO:8), or VISSHLGQ (SEQ ID NO:9), particularly where theantibody fragment is a Fab or F(ab′)₂. In another most preferredembodiment, the salvage receptor binding epitope is a polypeptidecontaining the sequence(s)(5′ to 3′): HQNLSDGK (SEQ ID NO:7), HQNISDGK(SEQ ID NO:8), or VISSHLGQ (SEQ ID NO:9) and the sequence: PKNSSMISNTP(SEQ ID NO:5).

B. Vectors, Host Cells and Recombinant Methods

The invention also provides isolated nucleic acid encoding an antibodyas disclosed herein, vectors and host cells comprising the nucleic acid,and recombinant techniques for the production of the antibody. Inaddition to recombinant production of the antibody, the nucleic acidencoding the antibodies disclosed herein may be used to inhibit cellsurface expression of the ErbB2 protein according to the teachings ofWO96/07321, published Mar. 14, 1996, for example. For example, theantibody may be a single chain Fv fragment provided in an expressionvector (such as a viral or plasmid vector), which vector is introducedinto a cell so as to bind to the ErbB2 protein intracellularly andthereby induce death of the cell.

For recombinant production of the antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression.

DNA encoding the monoclonal antibody is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibody). Many vectors are available. The vectorcomponents preferably include, but are not limited to, one or more ofthe following: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

The anti-ErbB2 antibody of this invention may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native anti-ErbB2 antibody signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeastsecretion the native signal sequence may be substituted by, e.g., theyeast invertase leader, a factor leader (including Saccharomyces andKluyveromyces α-factor leaders), or acid phosphatase leader, the C.albicans glucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable.

The DNA for such precursor region is ligated in reading frame to DNAencoding the anti-ErbB2 antibody.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Preferably, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Preferably, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theanti-ErbB2 antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti-ErbB2 antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the anti-ErbB2antibody nucleic acid. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, β-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tac promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgamo (S.D.) sequence operably linked to the DNAencoding the anti-ErbB2 antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Anti-ErbB2 antibody transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature, 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the anti-ErbB2 antibody of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theanti-ErbB2 antibody-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-ErbB2 antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E coli B, E coliX1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. Theseexamples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-ErbB2antibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-ErbB2antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophilamelanogaster(fruitfly), and Bombyx mori have been identified. A varietyof viral strains for transfection are publicly available, e.g., the L-1variant of Autographa californica NPV and the Bm-5 strain of Bombyx moriNPV, and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci.,383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-ErbB2 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

(viii) Culturing the Host Cells

The host cells used to produce the anti-ErbB2 antibody of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al. Meth. Enz., 58:44 (1979), Barnes etal., Anal. Biochem., 102:255 (1980), U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195;or U.S. Pat. Re. 30,985 may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

(ix) Purification of Anti-ErbB2 Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems arepreferably first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g. from about 0-0.25M salt).

C. Pharmaceutical Formulations

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide antibodies whichbind to EGFR, ErbB2 (e.g. an antibody which binds a different epitope onErbB2), ErbB3, ErbB4, or vascular endothelial factor (VEGF) in the oneformulation. Alternatively, or in addition, the composition may comprisea cytotoxic agent, cytokine or growth inhibitory agent. Such moleculesare suitably present in combination in amounts that are effective forthe purpose intended.

The active ingredients may also be entrapped in microcapsules prepared;for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

D. Non-therapeutic Uses for the Antibody

The antibodies of the invention may be used as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a Sephadex resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theErbB2 protein (or fragment thereof) to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the ErbB2 protein, which is boundto the immobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, that will release theErbB2 protein from the antibody.

Anti-ErbB2 antibodies may also be useful in diagnostic assays for ErbB2protein, e.g., detecting its expression in specific cells, tissues, orserum. Thus, the antibodies may be used in the diagnosis of humanmalignancies (see, for example, U.S. Pat. No. 5,183,884).

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can bepreferably grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.,Wiley-Interscience, New York, N.Y., Pubs., (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme preferablycatalyses a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73: 147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g. orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g. digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g. anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the anti-ErbB2 antibody need notbe labeled, and the presence thereof can be detected using a labeledantibody which binds to the ErbB2 antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of ErbB2 protein in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, andthereaftera second antibody binds to the analyte, thus forming aninsoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Preferably, the antibody is labelled with a radionuclide (such as ¹¹¹In,⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

E. Diagnostic Kits

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labelled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g. asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g. a block buffer or lysis buffer) and the like.The relative amounts of the various reagents may be varied widely toprovide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

F. Therapeutic Uses for the Antibody

It is contemplated that the anti-ErbB2 antibody of the present inventionmay be used to treat various conditions, including those characterizedby overexpression and/or activation of the ErbB2 receptor. Exemplaryconditions or disorders to be treated with the ErbB2 antibody includebenign or malignant tumors (e.g. renal, liver, kidney, bladder, breast,gastric, ovarian, colorectal, prostate, pancreatic, ling, vulval,thyroid, hepatic carcinomas; sarcomas; glioblastomas; and various headand neck tumors); leukemias and lymphoid malignancies; other disorderssuch as neuronal, glial, astrocytal, hypothalamic and other glandular,macrophagal, epithelial, stromal and blastocoelic disorders; andinflammatory, angiogenic and immunologic disorders.

The antibodies of the invention are administered to a mammal, preferablya human, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous administration of theantibody is preferred.

Other therapeutic regimens may be combined with the administration ofthe anti-ErbB2 antibodies of the instant invention. For example, thepatient to be treated with the antibodies disclosed herein may alsoreceive radiation therapy. Alternatively, or in addition, achemotherapeutic agent may be administered to the patient. Preparationand dosing schedules for such chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M.C. Perry,Williams & Wilkins, Baltimore, Md. (1992). The chemotherapeutic agentmay precede, or follow administration of the antibody or may be givensimultaneously therewith. The antibody may be combined with ananti-oestrogen compound such as tamoxifen or an anti-progesterone suchas onapristone (see, EP 616812) in dosages known for such molecules.

It may be desirable to also administer antibodies against other tumorassociated antigens, such as antibodies which bind to the EGFR, ErbB3,ErbB4, or vascular endothelial factor (VEGF). Alternatively, or inaddition, two or more anti-ErbB2 antibodies may be co-administered tothe patient. Sometimes, it may be beneficial to also administer one ormore cytokines to the patient. In a preferred embodiment, the ErbB2antibody is co-administered with a growth inhibitory agent. For example,the growth inhibitory agent may be administered first, followed by theErbB2 antibody. However, simultaneous administration or administrationof the ErbB2 antibody first is also contemplated. Suitable dosages forthe growth inhibitory agent are those presently used and may be lowereddue to the combined action (synergy) of the growth inhibitory agent andanti-ErbB2 antibody.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

G. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the anti-ErbB2 antibody.The label on, or associated with, the container indicates that thecomposition is used for treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringers solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

H. Deposit of Materials

The following hybridoma cell lines have been deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC):

Antibody Designation ATCC No. Deposit Date 7C2 ATCC HB-12215 Oct. 17,1996 7F3 ATCC HB-12216 Oct. 17, 1996 4D5 ATCC CRL 10463 May 24, 1990

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of deposit. The cell lines will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweenGenentech, Inc. and ATCC, which assures (a) that access to the cultureswill be available during pendency of the patent application to onedetermined by the Commissioner to be entitled thereto under 37 CFR §1.14and 35 USC §122, and (b) that all restrictions on the availability tothe public of the cultures so deposited will be irrevocably removed uponthe granting of the patent.

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedcell lines is not to be construed as a license to practice the inventionin contravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the antibodies deposited,since any antibody that is functionally equivalent is within the scopeof this invention. The deposit of material herein does not constitute anadmission that the written description herein contained is inadequate toenable the practice of any aspect of the invention, including the bestmode thereof, nor is it to be construed as limiting the scope of theclaims to the specific illustration that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

Example 1 Induction of Cell Death

Cell Lines.

The established human breast tumor cells BT474 and MDA-MB-231 (which areavailable from ATCC) were grown in minimum essential medium (Gibco,Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal bovineserum (FBS) (HyClone, Logan, Utah), sodium pyruvate, L-glutamine (2 mM),non-essential amino acids and 2× vitamin solution and maintained at 37°C. in 5% CO₂ (Zhang et al. Invas. & Metas. 11(4):204-215 (1991) andPrice et al. Cancer Res. 50(3):717-721 (1990)).

Antibodies.

The anti-ErbB2 IgG₁K murine monoclonal antibodies 4D5 and 7C2, specificfor the extracellular domain of ErbB2, were produced as described inFendly et al. Cancer Research 50:1550-1558 (1990) and WO89/06692.Briefly, NIH 3T3/HER2-3₄₀₀ cells (expressing approximately 1×10⁵ ErbB2molecules/cell) produced as described in Hudziak et al. Proc. Natl.Acad. Sci. (USA) 84:7159 (1987) were harvested with phospate bufferedsaline (PBS) containing 25 mM EDTA and used to immunize BALB/c mice. Themice were given injections i.p. of 10⁷ cells in 0.5 ml PBS on weeks, 0,2, 5 and 7. The mice with antisera that immunoprecipitated ³²P-labeledErbB2 were given i.p. injections of a wheat germ agglutinin-Sepharose(WGA) purified ErbB2 membrane extract on weeks 9 and 13. This wasfollowed by an i.v. injection of 0.1 ml of the ErbB2 preparation and thesplenocytes were fused with mouse myeloma line X63-Ag8.653. Hybridomasupernatants were screened for ErbB2-binding by ELISA andradioimmunoprecipitation. MOPC-21 (IgG1), (Cappell, Durham, N.C.), wasused as an isotype-matched control.

Analysis of Cell Cycle Status and Viability.

Cells were simultaneously examined for viability and cell cycle statusby flow cytometry on a FACSTAR PLUS™ (Becton Dickinson ImmunocytometrySystems USA, San Jose, Calif.). Breast tumor cells were harvested bywashing the monolayer with phosphate buffered saline (PBS), incubatingcells in 0.05% trypsin and 0.53 mM EDTA (Gibco) and resuspending them inculture medium. The cells were washed twice with PBS containing 1% FBSand the pellet was incubated for 30 minutes on ice with 50 μl of 400 μM7 amino actinomycin D (7AAD) (Molecular Probes, Eugene, Oreg.), a vitaldye which stains all permeable cells. Cells were then fixed with 1.0 mlof 0.5% paraformaldehyde in PBS and simultaneously permeabilized andstained for 16 hours at 4° C. with 220 μl of 10 μg/ml HOECHST 33342™ dye(also a DNA binding dye) containing 5% TWEEN 20™.

The data from 1×10⁴ cells were collected and stored using LYSYS II™software and analyzed using PAINT-A-GATE™ software (Becton Dickinson)(Darzynkiewica et al. Cytometry 13:795-808 (1992) and Picker et al. J.Immunol. 150(3):1105-1121 (1993)). The viability and percentage of cellsin each stage of the cell cycle were determined on gated single cellsusing 7AAD and Hoechst staining, respectively. (Cell doublets wereexcluded by pulse analysis of width vs. area of the Hoechst signal.)Cell numbers were determined using a hemocytometer.

DNA Synthesis.

Triplicate cultures of 8×10³ cells/well were plated in 96-well flatbottom plates, allowed to adhere overnight, then continuously incubatedin the presence or absence of anti-ErbB2 or control Ig for differentperiods of time. During the last 12 hours of culture, wells were pulsedwith 1 μCi ³H-thymidine (Amersham, Arlington, Va.).

Affinity of Binding to the Extracellular Domain of the ErbB2.

Radioiodinated anti-ErbB2 antibodies were prepared by the Iodogen method(Fracker et al. Biochem. Biophys. Res. Comm. 80:849-857 (1978)). Bindingassays were performed using monolayers of BT474 cells cultured in96-well tissue culture plates (Falcon, Becton Dickenson Labware, LincolnPark, N.J.). The cells were trypsinized and seeded in wells of 96-wellplates at a density of 10⁴ cells/well and allowed to adhere overnight.The monolayers were washed with cold culture medium supplemented with0.1% sodium azide and then incubated in triplicate with 100 μl of serialdilutions of ¹²⁵I-anti-ErbB2 antibodies in cold culture medium with 0.1%azide for 4 hours on ice. Non-specific binding was estimated by thepreincubation of each sample with a 100-fold molar excess ofnonradioactive antibodies in a total volume of 100 μl. Unboundradioactivity was removed by two washes with cold medium with 0.1%sodium azide. The cell-associated radioactivity was detected in a gammacounter after solubilization of the cells with 150 μl 0.1 M NaOH/well.The anti-ErbB2 binding constants (K_(d)) were determined by Scatchardanalysis.

Results.

The binding affinities of anti-ErbB2 antibodies (7C2 and 4D5) weredetermined by Scatchard analysis. The binding constants (K_(d)) were6.5×10⁻⁹ M (4D5) and 2.9×10⁹ M (7C2). Blocking experiments were carriedout using unlabelled antibodies followed by FITC-7C2. As shown in FIG.2, 4D5 reacts with a different epitope than 7C2.

The effect of these antibodies on the growth of the BT474 human breastcancer cells which overexpress ErbB2 was then investigated. FIG. 3Ashows the results of flow cytometric analysis of cells incubated with anisotype-matched control. 10-12% of the cells were dead and 28% of theviable cells were in the S-G₂-M phases of the cell cycle. Similarresults were obtained when the cells were incubated in medium alone.Treatment with 4D5 (FIG. 3B) induced a decrease in cell size as measuredby forward light scatter, a moderate increase in the proportion of deadcells (27.0%) and a marked decrease of viable cells in S-G₂-M (6.3%)with a concurrent increase of cells in G₀/G₁ (94%) as compared to thecontrol cells. Cell counts were reduced by 46.7%. Without being bound toany one theory, it appears that 4D5 induces primarily cell cycle arrest(CCA) in G₀/G₁ but that a significant proportion of cells also die. FIG.3C shows the results of incubating the BT474 cell with 50 μg/ml of 7C2.There was no change in forward light scatter of the residual viablecells, a marked increase in the proportion of dead cells (72%) and a 70%decrease in cell count compared to control cells (data not shown).Hence, viable cells were decreased by 85%. There was a slight reductionin cycling cells (21% vs. an average of 29% for controls) but because ofextensive cell death, it was difficult to distinguish CCA from apreferential loss of cycling cells. Hence, 7C2 and 4D5 affect cellsdifferently; 7C2 induces predominantly cell death while 4D5 inducespredominantly CCA. FIG. 3D shows the results of adding 50 μg/ml 7C2 and1 μg/ml 4D5 to BT474 cells simultaneously. There was no increase in theproportion of dead cells compared to cells treated with 7C2 alone (FIG.3C). However, the number of residual viable cells was reduced by anadditional 50%. In addition, there was a marked increase in cells havingboth permeable membranes and significantly degraded DNA (<1 X). Ananalysis of the small number of residual viable cells showed that therewas a similar reduction in cycling cells compared to cells treated with4D5 alone (FIG. 3D compared to FIG. 3B).

As an additional control, MDA-MB-231 breast cancer cells, which expressnormal levels of ErbB2 (Lewis et al. Cancer Immunol. Immunther.37:255-263 (1993)), were treated with 405 or 7C2. As compared to thecontrol, neither antibody affected the growth of these cells (27-28% ofviable cells in S-G₂-M and 12-13% dead cells).

The additive effects of both antibodies was dearly demonstrated when asuboptimal dose of 4D5 (0.05 μg/ml) was used. FIG. 4A shows thatthymidine incorporation was reduced by 22.3% and 23% in cells treatedwith 7C2 and 4D5 respectively, and by 58% in cells treated with 4D5 and7C2. An additional experiment utilizing 20 μg 7C2 and 0.1 μg 4D5 gavesimilar results, i.e., thymidine incorporation was reduced by 41%, 25%and 72% in cells treated with 7C2 alone, 4D5 alone, or 4D5 plus 7C2,respectively. In FIG. 4B, the viable cell counts are shown. A total cellcount was determined and FACS analysis was used to establish the numberof viable cells. Viable cell counts were reduced by 64%, 29% and 84% incells treated with 7C2, 4D5 or the combination, respectively, whencompared to untreated controls.

Example 2 Induction of Apoptosis

Materials and Cell Culture.

All tumor cell lines were obtained from the American Type CultureCollection (Rockville, Md.). Cells were cultured in Dulbecco's ModifiedEagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with 10%heat-inactivated fetal bovine serum (FBS) (Hyclone) and 2 mML-glutamine. Human mammary epithelial cells (HMEC) were obtained fromClonetics and grown in MEGM (mammary epithelial growth medium,Clonetics) containing bovine pituitary extract. Biochemicals used were:annexin V-FTIC (BioWhittaker, Inc.), propidium iodide (PI, MolecularProbes, Inc.), and HOECHST 33342™ (Calbiochem). Anti-ErbB2 monoclonalantibodies (MAbs) were produced as described in Fendly et al. CancerResearch 50:1550-1558 (1990) and WO89/06692 (see Example 1 above). Theanti-ErbB2 MAbs tested are designated: 4D5, 7C2, 7F3, 3H4, 2C4, 2H11,3E8, and 7D3. The isotype-matched control MAb 1766 is directed againstthe herpes simplex virus (HSV-1) glycoprotein D.

Flow Cytometry Experiments for Measuring Induction of Apoptosis.

Cells were seeded at a density of 3×10⁶ per dish in 100×20 mm dishes andallowed to attach overnight. The medium was then removed and replacedwith fresh medium alone or medium containing 10 μg/ml of the appropriateMAb. For most experiments, cells were incubated for a 3 day time period.For time course studies, cells were treated for 0.25, 0.5, 1, 2, 24, 72,96 hr, 7 or 10 days. MAb concentrations used in the dose-responseexperiments were 0.01, 0.1, 1 and 10 μg/ml. Following each treatment,supernatants were individually collected and kept on ice, monolayerswere detached by trypsinization and pooled with the correspondingsupernatant. Cells were then centrifuged at 1200 rpm for 5 minutes at 4°C., the pellet resuspended in 3 ml ice cold Ca²⁺ binding buffer (10 mMHepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mmstrainer-capped 12×75 tubes (1 ml per tube, 3 tubes per treatment group)for removal of cell aggregates. Each group of 3 tubes then receivedannexin V-FTIC (1 μg/ml) or PI (10 μg/ml) or annexin V-FTIC plus PI.Samples were analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™CellQuest software (Becton Dickinson). For cell cycle analysis, cellswere incubated with 9 μg/ml HOECHST 33342™ for 2 hr at 37° C., thenanalyzed on an EPICS ELITE™ flow cytometer (Coulter Corporation) usingMODFIT LT™ software (Verity Software House).

Serum-deprivation experiments were performed in the following way. BT474breast tumor cells were seeded in culture medium at a density of 5×10⁶per dish in 100×20 mm dishes. The following day, the medium was replacedwith medium containing 0.1% FBS and the cells were incubated for 3 days.Cells then received 10 μg/ml of MAb 7C2 or 4D5 in fresh mediumsupplemented with 0.1% FBS. After a 3 day incubation, analyses ofannexin V binding, PI uptake and cell cycle progression were performedas described above. In order to compare growth of serum-starved cells tonon-deprived cells, separate dishes of BT474 cells, incubated in mediumsupplemented with 10% FBS for all time points, were studied in parallel.

Detection of DNA Ladder Formation.

For measuring internucleosomal fragmentation of DNA, BT474 breast tumorcells were plated and treated for 3 days with 10 μg/ml MAb 4D5 or 7C2 asdescribed above. DNA was extracted, ³²P-end-labeled, and run on a 2%agarose gel containing 5 μg/ml ethidium bromide. The gel was then driedand exposed to Kodak film. The formation of DNA ladders, a hallmark ofapoptosis, was observed in BT474 breast tumor cells treated with 10μg/ml MAb 7C2 or MAb 4D5 for 3 days.

Electron Micrography Studies.

BT474 cells were treated with 10 μg/ml MAb 7C2 for 3 days, then fixed in1.25% formaldehyde/1% glutaraldehyde in 0.1M cacodylate buffer.Post-fixation was performed in 2% osmium tetroxide in cacodylate buffer.The fixed cells were then end-block stained in uranyl acetate,dehydrated in graded. concentrations of ethanol, and embedded in Eponet.Sections were cut utilizing a microtome and observed under a PhilipsCM12™ electron microscope. Highly shrunken cells displaying nuclear andcytoplasmic condensation, typical of apoptotic cells, were observedafter treatment with MAb 7C2. The apoptotic cells eventually becomephagocytosed by underlying cells.

The results of the experiments performed are shown in FIGS. 5-14.Certain anti-ErbB2 MAbs induce apoptosis in human tumor cell lines whichoverexpress ErbB2 as evidenced by electron microscopy, annexin-Vbinding, cell cycle analysis of DNA content, DNA laddering andtime-lapse videomicrography. Anti-ErbB2 MAbs 7C2 and 7F3, whichrecognize the same epitope on the ErbB2 extracellular domain, displaythe most potent pro-apoptotic effects. Anti-ErbB2 MAb 4D5, whichrecognizes a different ErbB2 epitope, induces a small amount ofapoptosis in addition to its potent reduction in proliferation.Induction of apoptotic cell death by 7C2 or 4D5 appears to beindependent of cell cycle. Inhibition of growth, either by serumdeprivation or by treatment with 4D5, followed by treatment with 7C2 canresult in complete cell death of the culture.

Example 3 Apoptosis of Ovarian Cells and Combination Treatment

Methods. SKOV3 ovarian cancer cells were seeded at a density 10⁶ perdish in 20×100 mm dishes and allowed to attach for 2-3 days. The mediumwas then removed and replaced with fresh medium alone or mediumcontaining the appropriate anti-HER2MAb. For studies on treatment withsingle MAb, cells were incubated with 10 μg/ml MAb 7C2 or 4D5 for 3days. For antibody combination treatments, BT474 cells were treatedfirst for 24 hr with 0.25 or 0.5 μg/ml MAb 7C2. Following thistreatment, 10 μg/ml MAb 4D5 was added and the cells were incubated for 3more days. Following each treatment, supernatants were individuallycollected and kept on ice, monolayers were detached by trypsinizationand pooled with the corresponding supernatant. Cells were thencentrifuged at 1200 rpm for 5 min at 4° C., the pellet resuspended inice cold Ca²⁺ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mMCaCl₂) and aliquoted into 35 mm strainer-capped 12×75 tubes. Cells werestained with 1 μg/ml annexin V-FITC and 10 μg/ml propidium iodide (PI)and analyzed on a FACScan flow cytometer using FACSCONVERT CELLQUEST™software (Becton Dickinson).

Induction of Apoptosis in Ovarian Cells.

The percent of remaining viable (annexin V negative/PI negative) cellsafter 3 days of treatment with MAb 7C2 was reduced by 48% (3.8 foldincrease in annexin V binding) in the SKOV3 ovarian carcinoma line (FIG.15).

Combination Treatment.

Induction of apoptosis by the sequential addition of MAbs 7C2 and 4D5leads to additive effects compared to either antibody alone (FIG. 16).Treatment of BT474 breast tumor cells with 0.5 μg/ml MAb 7C2 followed by10 μg/ml MAb 4D5 results in a reduction in viable (annexin V negative/PInegative) cells to 12%, compared to 68% or 29.1% with MAb 4D5 or 7C2alone, respectively. This additive effect is also seen with a suboptimaldose of MAb 7C2, where treatment with 0.25 μg/ml 7C2 plus 10 μg/ml 4D5leads to a decrease in viable cells to 37.5%, compared to 68% for MAb4D5 alone and 77% for 7C2 alone. Simultaneous addition of MAbs 7C2 and4D5 or addition of MAb 4D5 prior to 7C2 does not appear to lead toadditive effects on cell death. Thus, without being bound by any onetheory, the sequential application of MAb 7C2, then MAb 4D5, may beimportant for achieving an enhanced apoptotic effect.

Example 4 In Vivo Effects of Anti-HER2 Antibodies

A xenograft model of HER2 overexpressing human breast cancer wasestablished to assess the effect of anti-HER2MAbs in relation toHER2-overexpressing tumors. The model uses the BT474 cell line selectedin vivo for growth in nude mice, BT474M1. Tumor bearing mice weretreated twice weekly with monoclonal antibodies, 4D5, 7C2 or 7F3 orcombinations of 4D5 with 7C2 or 7F3. Tumor growth was assessed bymeasuring tumor size twice weekly. The 4D5 monoclonal antibody hadsignificant growth inhibitory effects on HER2 overexpressing xenografttumors from the BT474M1 cell line. These results are similar topreviously published results. The monoclonal antibodies 7C2 and 7F3 hadmodest growth inhibitory effects on their own, at the doses tested, andsignificantly enhanced the growth inhibitory effect of 4D5. None of theantibodies exhibited any toxic effect on the animals.

Animal Model.

NCR.nu/nu mice (homozygous females, 4 weeks of age) were implantedsubcutaneously with 0.72 mg sustained release 17βestradiol pellets tosupport the growth of tumor. Animals were inoculated by subcutaneousinjection with 5 million BT474M1 tumor cells in MATRIGEL™ 24 hours afterestrogen implantation. Animals were monitored daily for well being andtumors were measured twice weekly. Reports on tumor measurements weresupplied as they were collected. Animals were weighed weekly to assesstoxicity during the study. One hundred five (105) animals wereinoculated and all animals were evaluated in the study.

Treatment Protocol.

Animals were randomized to one of 7 treatment groups (15 animals pergroup). Treatment was initiated 6 days after inoculation of tumors.Tumor sizes for all animals and mean tumor sizes for each of thetreatment groups were examined prior to beginning treatment to ensureconsistency between groups. Treatment groups were:

1) Vehicle control injection—100 μl by intraperitoneal (IP) injectiontwice weekly2) Irrelevant antibody (agp120) (isotype matched)—10 mg/kg in 100 μl IPtwice weekly3) MAb 7C₂₋₁₀ mg/kg in 100 μl by IP injection twice weekly4) MAb 7F3—10 mg/kg in 100 μl by IP injection twice weekly5) MAb 4D5—10 mg/kg in 100 μl by IP injection twice weekly6) MAb 7C2 (10 mg/kg)+MAb 4D5 (10 mg/kg)—in 100 μl by IP injectionweekly7) MAb 7F3 (10 mg/kg)+MAb 4D5 (10 mg/kg)—in 100 μl by IP injectionweekly

All treatment groups were treated twice weekly for a total of 10treatments by intraperitoneal injection. Treatment groups 1-4 wereeuthanized at this point due to large tumor size. Treatment groups 5, 6and 7 were continued and received a total of 16 antibody treatments.Throughout the study, animals were monitored daily for well being, twiceweekly for tumor measurements and weekly for body weights. Individualanimals data and mean data by treatment group was supplied as it becameavailable.

Termination of Experiment.

After conclusion of treatment, animals in treatment groups 1-4 wereeuthanized due to some large tumor sizes within these treatment groups.Tumor volume was not allowed to exceed 4 gms (4,000 mm³). No animalswere observed with significant weight loss and weight loss greater than15% loss of body weight from the weight at onset of treatment was neverobserved. At the conclusion of the experiment, all animals wereeuthanized.

Results.

The monoclonal antibodies, 4D5, 7C2 and 7F3, directed against the HER2growth factor receptor, were used in a mouse xenograft model whichoverexpresses the HER2 receptor. Antibodies were used alone and incombinations of growth inhibiting antibody (4D5) with apoptoticantibodies (7C2 and 7F3). The apoptotic antibodies (7C2 and 7F3) had agrowth inhibitory effect early on in the study that was lost at latertime points (FIG. 17). The growth inhibitory antibody, 4D5, had a markedgrowth inhibitory effect throughout the study, as has been reported inprevious studies. The combination of 4D5 with either 7C2 or 7F3potentiated the growth inhibitory effect significantly, with 4D5/7C2being the best combination. There was one complete remission in the 4D5alone treatment group and one complete remission in the 4D5/7C2treatment group. The antibody control group (anti-gp120 MAb) wasequivalent to the saline treated control group. Body weights initiallyincreased after tumor inoculation, then were maintained through theremainder of the study. None of the antibodies exhibited any toxiceffect on the animals.

1-41. (canceled)
 42. A method of treating cancer comprisingadministering to a human subject with cancer characterized byoverexpression of the ErbB2 receptor a first antibody that binds Domain1 (SEQ ID NO: 1) of ErbB2 and thereby induces cell death in cancer cellsoverexpressing ErbB2, or an antigen-binding fragment of said firstantibody, and a second antibody that binds ErbB2 at a different domainthan Domain 1 and inhibits growth of cancer cells overexpressing ErbB2,or an antigen-binding fragment of said second antibody.
 43. The methodof claim 42, wherein the first antibody binds epitope 7C2/7F3 (SEQ IDNO: 2) of ErbB2.
 44. The method of claim 42, wherein the first antibodycomprises CDRs of antibody 7C2 or 7F3.
 45. The method of claim 42,wherein the first antibody induces apoptosis in said cells.
 46. Themethod of claim 42 wherein the second antibody comprises CDRs ofantibody 4D5.
 47. The method of claim 42, wherein the first antibodycomprises CDRs of antibody 7C2 or 7F3 and the second antibody comprisesCDRs of antibody 4D5.
 48. The method of claim 42, wherein the first andthe second antibodies are chimeric, humanized or human.
 49. The methodof claim 42, wherein the cancer is breast cancer.
 50. The method ofclaim 49, wherein administration is performed intravenously as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.
 51. Themethod of claim 50 wherein administration is performed intravenously.